No abstract

With the increasing number of patients with hypertensive nephropathy worldwide, it has posed a major threat to health and studies on its treatment and pathogenesis are imminent. The present study investigated the mechanism through which microRNA (miR)-98-5p in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) is involved in the repair of angiotensin II (Ang II)-induced injury of rat primary renal kidney cells (PRKs). After isolation of rat renal cortical sections, PRKs were isolated by density gradient centrifugation and identified by immunofluorescence staining. Transmission electron microscopy identifies successful separation of Mvs. An in vitro cell injury model was constructed using Ang II. The Gene Expression Omnibus was used to analyze the differentially expressed genes between diabetic rats and normal rats, and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the signaling pathways involved in these differentially expressed genes. Reverse transcription-quantitative PCR was used to analyze the effect of EPC-MVs on the expression of miRNA induced by Ang II, and the levels of target genes and signaling pathway-related proteins involved were analyzed by western blot. luciferase was used to detect the targeted binding of miR-98-5p to insulin-like growth factor 1 receptor (IGF1R). Enzyme-linked immunosorbent assay was used to analyze the effect of EPC-MVs on Ang II-induced oxidative stress and inflammation levels on PRKs. Cell Counting Kit-8 was used to analyze the effect of EPC-MVs on the cell viability of PRKs induced by Ang II. The results showed that treatment of PRKs with Ang II decreased cell viability, whereas oxidative stress and inflammation were increased. However, EPC-MVs alleviated Ang II-induced damage of the PRKs. During this process, the Ang-II-induced downregulation of miR-98-5p was reversed by EPC-MVs, so miR-98-5p may be a key factor regulating the action of EPC-MVs. Dual-luciferase assay confirmed that miR-98-5p targets IGF1R. It was subsequently demonstrated that EPC-MVs overexpressing miR-98-5p promoted phosphorylation of PI3K/Akt/endothelial nitric oxide synthase (eNOS), and inhibited the oxidative stress and inflammation in PRKs, which were reversed by the overexpression of IGF1R. In conclusion, the results of the present study demonstrated that EPC-MVs with high expression of miR-98-5p can activate the PI3K/Akt/eNOS pathway by regulating IGF1R, as well as protect PRKs from Ang II-induced oxidative stress, inflammation and inhibition of cell viability.

Background. This study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs). Methods. Gene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry. Results. A total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206. Conclusion. By upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.